Preparative Denaturing Isoelectric Focusing for Enhancing Sensitivity of Proteomic Studies

  • Antonio Serna-Sanz
  • Greg Rairdan
  • Scott C. Peck
Part of the Methods in Molecular Biology book series (MIMB, volume 354)


Substantial evidence implicates important roles for both protein phosphorylation and protein degradation in regulation of plant defense responses. Therefore, interest is growing in applying proteomics techniques to investigate these posttranscriptional changes. We have found, however, that most proteins of interest are not visible on two-dimensional (2D) gels without previous prefractionation. This chapter describes the use of preparative denaturing isoelectric focusing to enrich for proteins of specific isoelectric points before separation by 2D gels. This method significantly increases the sensitivity of 2D gelbased comparisons.

Key Words

Proteomics 2D gel preparative isoelectric focusing phosphoproteomics 


  1. 1.
    Nuhse, T. S., Boller, T., and Peck, S. C. (2003) A plasma membrane syntaxin is phosphorylated in response to the bacterial elicitor flagellin. J. Biol. Chem. 278, 45,248–45,254.PubMedCrossRefGoogle Scholar
  2. 2.
    Peck, S. C., Nuhse, T. S., Hess, D., Iglesias, A., Meins, F., and Boller, T. (2001) Directed proteomics identifies a plant-specific protein rapidly phosphorylated in response to bacterial and fungal elicitors. Plant Cell 13, 1467–1475.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Antonio Serna-Sanz
    • 1
  • Greg Rairdan
    • 2
    • 3
  • Scott C. Peck
    • 1
  1. 1.The Sainsbury LaboratoryJohn Innes CenterNorwichUK
  2. 2.Boyce Thompson Institute for Plant ResearchCornell UniversityIthaca
  3. 3.The Sainsbury LaboratoryJohn Innes CenterNorwichUK

Personalised recommendations